This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Glycosphingolipids and gangliosides participate in diverse biological processes, and their biological roles are dependent on the structures of both the oligosaccharide and the ceramide portions. Here, vibrationally cooled (VC)MALDI-FTMS was used for the detection of labile species followed by their efficient fragmentation by SORI-CAD and IRMPD. GM1 and GD1a gangliosides serve as trafficking receptors for cholera toxin and the related LTIIb toxin, respectively. We assume that GD1a ganglioside of human intestinal cells is not associated with lipid rafts due to its ceramide structural variation, which prevents endocytosis of the LTIIb-GD1a complex. The toxin receptors' ganglioside structures were evaluated as a moderator of this function, including ceramide chain length, level of saturation and hydroxylation, as well as glycan composition. To analyze these molecules, our previously developed method of direct coupling of TLC plates with VC-MALDI-FTMS was used. This allows direct TLC-MALDI-FTMS without adversely affecting the FT high resolution and mass accuracy by the surface irregularity of the TLC plate. Collisional cooling is necessary for stabilization and detection of intact gangliosides. Our earlier reports have described ganglioside purification from polarized intestinal epithelial cell line T-84 and monkey kidney Vero cells and functional studies on the mechanism of toxin biology. The samples were MALDI-desorbed directly off TLC plate surfaces with ~0.2 mm sampling steps. Fragmentation was subsequently performed by SORI-CAD and IRMPD. Both sialylated and highly fucosylated glycosphingolipids were observed. GC/MS analysis of released lipids and glycans were consistent with these observations. A fraction, which could be a protein fragment, that does not migrate on TLC is now being studied, since it also binds with the toxin. Two manuscripts are in preparation.